CHOLESTEROL, STEROIDS, AND RELATED MOLECULES

Peter D'Eustachio – December 4 - 5, 2006

Objectives. Understand the overall pathways of sterol uptake, synthesis, transport, and
utilization and their regulation; be able to explain why a deficiency of LDL receptors
leads to elevated blood cholesterol. Understand the rationale for pharmacological inter-
ventions to lower cholesterol levels. Understand the overall pathways, regulation and tis-
sue specificity of steroid hormone biosynthesis; be able to use these features of the proc-
ess to explain the biochemical consequences of defects in specific synthetic enzymes.
Cholesterol structure. Cholesterol contains 27 carbon atoms arranged into four fused
rings and a hydrocarbon tail.

The molecule is extremely hydrophobic. It is an essential component of cell membranes,

and is the precursor for the synthesis of several important molecules. These include bile
acids (needed for the digestion and absorption of dietary fat), vitamin D (needed for cal-
cium uptake and utilization), and steroid hormones (needed for regulation of ion balance,
metabolism, and sexual differentiation).
Overview of cholesterol metabolism. Cholesterol can be obtained from the diet, or can
be synthesized, primarily in the liver and adrenal gland, from acetyl-CoA. Its main fates
are export to peripheral tissues for membrane biogenesis and conversion to bile salts in
the liver. Synthesis of steroid hormones, while essential, accounts for less than 10% of
cholesterol utilization. Cholesterol is lost from the body as bile salts that are not reab-
sorbed, in the membranes of dead epithelial cells sloughed off from body surfaces, and as

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steroid hormone derivatives excreted in the urine and feces. There is no metabolic path-
way to use cholesterol for the production of energy.

Cholesterol biosynthesis. Cholesterol is synthesized from acetyl-CoA, with NADPH +

H+ as a source of reducing equivalents and ATP as a source of energy, in a sequence of
approximately 30 reactions. Cholesterol is not synthesized by plants or bacteria, so a diet
rich in vegetable products is low in cholesterol.

Most cells in the body are capable of carrying out these reactions, but the bulk of choles-
terol biosynthesis occurs in the liver. Substantial amounts are also made in the adrenal
gland and gonads (where it serves as a precursor for steroid biosynthesis).

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Synthesis begins in the cytosol, with the condensation of acetoacetyl-CoA and acetyl-
CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This reaction is catalyzed
by a cytosolic isoform of HMG-CoA synthase, distinct from the mitochondrial isoform
used in ketone body synthesis. The next reaction, the conversion of HMG-CoA to meva-
lonate catalyzed by HMG-CoA reductase , is the committed step of cholesterol biosyn-
thesis. This step is also tightly regulated: both sterols (end products of the pathway) and
glucagon trigger signaling cascades that cause phosphorylation and inactivation of the
enzyme; insulin triggers its dephosphorylation and activation. The major form of regula-
tion of HMG-CoA reductase, however, is at the level of transcription.
A key intermediate in cholesterol biosynthesis is farnesyl pyrophosphate, which can be
covalently attached to proteins. Many proteins that are attached to the cytosolic face of
the plasma membrane, including RAS GTPases, are held there via farnesyl groups.
About 20 reactions are needed to convert squalene to cholesterol; these occur in the en-
doplasmic reticulum.

(modified from Horton et al. (2002) J Clin Invest 109:1125-1131)

Transcriptional regulation of cholesterol biosynthesis. The major factor controlling
the rate of cholesterol biosynthesis and linking it to cholesterol uptake from the circula-
tion is transcriptional regulation of HMG CoA synthase, HMG CoA reductase, and the

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LDL receptor. This regulation is mediated by the transcription factor, SREBP (sterol re-
sponse element binding protein). SREBP is produced as a membrane-bound protein at-
tached to the endoplasmic reticulum of the cell via a second protein, SCAP. In choles-
terol-starved cells SREBP:SCAP is transported to the Golgi apparatus where it is cleaved
sequentially by two proteases (designated S1P and S2P) that release the bHLH fragment
of SREBP into the cytosol. bHLH then enters the nucleus where it activates several key
genes of sterol metabolism, including the HMG CoA reductase gene.
Cholesterol does not interact directly with the SCAP:SREBP complex, but rather with a
third protein, INSIG. The INSIG:sterol complex traps the proteins responsible for trans-
porting SREBP:SCAP to the Golgi apparatus and thus immobilizes SCAP:SREBP in the
endoplasmic reticulum. Reduced cholesterol levels lead to dissociation of INSIG:sterol,
allowing SCAP:SREBP to migrate to the Golgi complex where the signaling domain of
SREBP is released by proteolysis to travel into the nucleus.
Another complication not shown in the figure is that there are three SREBP isoforms,
which differ in the efficiencies with which they bind to the various SREBP-regulated
genes. The different isoforms are expressed at different levels in different tissues, sug-
gesting that regulation of sterol metabolism may be tissue-specific.
Note that all of these regulatory mechanisms operate at the level of individual cells, sens-
ing and adjusting cholesterol levels at the single-cell level.
Bile acids and salts. Bile acids are cholesterol derivatives with two (commonly) or one
(less commonly) addi-
tional hydroxyl groups,
whose hydrocarbon tail
has been shortened and
oxidized to form a car-
boxylate group. The addi-
tional hydrophilic groups
increase these molecules’
solubility in water, making
them effective detergents.
Conjugation of the car-
boxylate group with gly-
cine or taurine further in-
creases their solubility.
These conjugates are
called bile “salts”.
As indicated by the dashed
arrow, a variant of these
reactions may also provide
a means to mobilize and remove excess cholesterol from neural tissue.

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The rate of bile acid syn-
thesis is determined
largely by transcriptional
regulation of 7-alpha-
hydroxylase, the first
enzyme in the pathway
from cholesterol to bile
acids. Transcription of
the 7alpha-hydroxylase
gene is positively regu-
lated by SREBP, as de-
scribed above. It is also
positively regulated by
LHR. Negative regula-
tion by high levels of
bile acids is achieved
indirectly. Bile acids can form a complex with the LXR transcription factor, enabling its
movement into the nucleation and transcriptional activation of the SHP gene. SHP pro-
tein in turn can form a heterodimer with LXR which, instead of acting as a transcriptional
activator, acts as a transcriptional repressor for 7alpha-hydroxylase. In effect, the normal
function of SHP protein is analogous to that of a dominant negative mutant protein.
These regulatory events are outlined in the drawing. Note that the LXR signaling process
exactly parallels the positive signaling process discussed earlier for glucocorticoid hor-
mone receptors.
Pharmacological interventions to manage cholesterol levels. Normal sterol synthesis
and re-utilization is controlled at
multiple levels by the body to as-
sure a sufficient supply of these
essential molecules. To achieve
substantial reductions of serum
cholesterol, several targets are at-
tacked simultaneously.
Statins like “Lovastatin” are potent
specific competitive inhibitors of
HMG CoA reductase, and block de
novo cholesterol biosynthesis.
Resins like cholestyramine bind
bile salts in the gut, preventing
their reutilization.
Ezetimbe interferes with cholesterol uptake from the gut, possibly by modifying the ac-
tivity of ABC transporters.
The resulting fall in intracellular sterol levels causes increased production of HMG CoA
reductase, 7alpha hydroxylase, and LDL receptor proteins, the net effect of which is to
draw cholesterol into the liver for conversion to bile acids and excretion.

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Vitamin D synthesis.
7-dehydrocholesterol,
an immediate precur-
sor of cholesterol on
the de novo biosyn-
thetic pathway, is con-
verted to vitamin D in
a reaction driven by
exposure to UV light
in the skin, and enzy-
matic reactions that
occur in the liver and
kidney.

Steroid hormones. This diagram is an overview of the process of steroid hormone bio-
synthesis in the human body, highlighting a key regulated step, several enzymes useful
for understanding normal tissue specificity of these events, and several key products.

In any given tissue, the amount of steroid synthesis is controlled by the availability of
cholesterol in the mitochondrial matrix (determined in turn by the abundance of StAR, a
cholesterol transporter protein in the inner mitochondrial membrane), and the identities of
the steroids synthesized are determined by the presence or absence of specific enzymes.
Although these molecules are hydrophobic, both the end products and many intermedi-

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ates can leave the cells in which they are made and circulate as complexes either with se-
rum albumin or with specific sterol-binding globulin proteins.
These general principles can be used to rationalize two examples of normal tissue-
specific hormone synthesis, and one pathological state, congenital adrenal hyperplasia
due to 21 alpha hydroxylase deficiency.
Cortisol biosynthesis. Cortisol is
a glucocorticoid hormone, acti-
vating the transcription of genes
that mediate the body’s response
to stress. In the Zona reticularis
and Zona fasciculata of the adre-
nal cortex, ACTH stimulates syn-
thesis of StAR, leading to in-
creased cortisol biosynthesis.
Elevated cortisol levels, in turn,
inhibit ACTH release, StAR pro-
tein levels fall, and cortisol syn-
thesis is reduced as discussed in
connection with amino acid me-
tabolism and the body’s response
to stress.
Aldosterone biosynthesis. Aldosterone, a mineralocorticoid, promotes transcription of
genes in the kidney whose protein products mediate Na+ reuptake. These reactions occur
in the Zona glomerulosa of the adrenal cortex. Here, StAR protein levels are increased in
response to angiotensin II, a peptide hormone that signals reduced Na+ levels in the
blood. StAR gene transcription is blocked in response to atrial natriuretic factor, a signal
of elevated Na+ levels. The StAR protein has a short half-life, so transcriptional regula-
tion effectively controls aldosterone levels under normal conditions. Although aldoster-
one biosynthesis and cortisol biosynthesis both require an 11 beta hydroxylation reaction,
these reactions are catalyzed by
two different, tissue-specific iso-
forms of 11 beta hydroxylase.
The B2 isoform, expressed in the
Zona glomerulosa, catalyzes the
conversion of 11-deoxycortisone
to cortisone (as well as the fol-
lowing reaction) but has no activ-
ity on 11-deoxycortisol. Thus,
progesterone generated in the
Zona glomerulosa is converted
specifically into aldosterone,
while the same intermediate in the
Zona reticularis and Zona fascicu-
lata is channeled instead into cor-
tisol production.

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Congenital Adrenal Hyperplasia. A mutation affecting a single enzyme, 21 alpha hy-
droxylase, has diverse physiological effects which can be rationalized on the basis of the
themes used to explain normal regulation of steroid hormone synthesis and its tissue
specificity. In the adrenal cortex, loss of 21 alpha hydroxylase activity blocks the synthe-
sis of both glucocorticoids (corti-
sol) and mineralocorticoids (al-
dosterone). To the extent that
progesterone is generated, it will
instead all be converted to andros-
tenedione, which in turn can leave
the adrenal cortex, bind carrier
proteins in the blood, and be
transported to skin or the male
gonad to be converted to testos-
terone. Because cortisol synthesis
is reduced, ACTH levels will re-
main high, StAR protein produc-
tion in cells of the adrenal cortex
will remain high, and progester-
one synthesis will proceed
briskly.